Understanding the complex spatio-temporal interplay of nanornaterials used as therapeutics at the cellular to the molecular level is required for designing nanoparticle based therapeutics and biomarkers for many diseases. To study these interactions, fluorescent labeling is commonly used for both in vivo cellular imaging and in vitro assay detection. Conventional fluorescence imaging involves single photon excitation of higher energy to emit lower energy light, a process known as down conversion. Organic dyes, metal and semiconductor nanoparticles are widely used as fluorophores to follow ligands into the cells and within tissue in animals as described in Michalet X, Pinaud F, Bentolila L, Tsay J, Doose S, Li J, Sundaresan G, Wu A, Gambhir S, Weiss S. Quantum Dots for Live Cells, in Vivo Imaging, and Diagnostics. Science 2005; 307, pp. 538-544. The fluorophores suffer from auto-fluorescence from biological tissues, photobleaching, low signal-to-noise ratio, potential damage to DNA and cell death as described in Green M, Howman E. Semiconductor quantum dots and free radical induced DNA nicking. Chem Commun 2005, pp. 121-123 and in Riegler J, Nann T. Application of luminescent nanocrystals as labels for biological molecules. Anal Bioanal Chem 2004, 379, pp. 913-919.
Up conversion phosphors (UCPs) capable of converting near infrared (NIR) radiation into shorter wavelengths through a multi-photon process, offers an alternative with minimal photo damage and auto-fluorescence due to the noninvasive nature of light as described in Auzel F. Upconversion and Anti-Stokes Processes with f and d Ions in Solids. Chem Rev 2004, pp. 139-173. Since up conversion occurs within the host crystal and is therefore less affected by the chemical and biological environments, and allows synthesis of materials without the loss of surface chemical reactivity.
All these favorable properties indicate that there is a real potential for the development of up conversion phosphors in the analysis of biological samples, especially for fluorescent imaging in vivo. Up conversion phosphors contain a sensitizer ion which absorbs the near infrared photons and transfers the absorbed energy sequentially to excite an emitter ion into a state which then emits in the visible. Lanthanide ions are particularly suitable candidates for up conversion processes because of their energy level structure providing many intermediate levels with favorable spacings and long-lived excited states. Yb3+ ions have been widely used as sensitizer as Yb3+ ions exhibit a relatively large absorption cross section in the near infrared region allowing excitation with laser diodes.
Metal fluorides, oxysulfides and phosphates are the matrices widely used to study the up conversion process and only a few reports are available with oxides. However, in order to have biocompatibility, a silica layer has been often coated on the surface of up conversion phosphors. Among the rare earth oxides, cerium oxide nanoparticles (CNPs) are shown to be biocompatible and exhibit regenerative antioxidant properties.
Earlier studies by co-inventors indicate that cerium oxide nanoparticles offer carbonic anhydrase inhibition, protection of primary cells from the detrimental effects of radiation therapy, prevention of retinal degeneration induced by intracellular peroxides, and neuroprotection to spinal cord neurons and have radical scavenging properties. By tailoring the surface potential or conjugating with targeting agents, specific uptake of cerium oxide nanoparticles in cells can be achieved. Although enhanced cellular uptake has been optimized based on surface characteristics, the mechanism of interaction, absorption and metabolism of these nanomaterials under in vivo condition is poorly understood.
The interaction of cerium oxide nanoparticles with cells and tissues needs to be well established for biomedical applications. Through real time imaging of cell-nanoparticle interactions it is possible to better understand the complex processes and conditions that facilitate or inhibit the uptake and release of materials into the cytosol or other intracellular targets. But, cerium oxide nanoparticles show weak emission characteristics in visible region, inhibiting the direct use of these materials for imaging purposes. One of the approaches to enhance the emission of cerium oxide nanoparticles is by doping with europium which has strong emission in the visible region. But, combining the non-toxic, biocompatible properties of cerium oxide nanoparticles with up conversion dopants would be a novel approach to optimize the emission properties. However, it is important to assess the interaction between the nanoparticles and cells since the biocompatibility and cellular uptake properties determine the therapeutic and imaging applications.
The present invention focuses on the formulation of co-doped cerium oxide nanoparticles with Yb3+ sensitizer and mainly Er3+ as an emitter. These co-doped, annealed cerium oxide nanoparticles will hereafter be referred to as up conversion nano ceria (UNC). In order to demonstrate the emission wavelength tunability, the emitter ion was changed to Ho3+ or Tm3+. The physical and optical properties of up conversion nano ceria and characteristics of these novel materials in various cells (A549, WI-38, HUVEC) were investigated by the co-inventors. In order to assess the antioxidant properties, catalase mimetic activity to catalyze the decomposition of hydrogen peroxide into water and oxygen was studied. The influence of nanoparticle interaction on the cell viability of normal lung fibroblasts (CCL-135) and cancerous cells (CRL-5803) were analyzed.